The thirteenth-century human occupants of the Promontory caves, Utah, distinguished themselves from surrounding Fremont populations by being successful hunting specialists of bison in a peripheral region for that species. The hunters’ success is evident from the abundance of faunal remains excavated from the caves. The dry cave conditions preserved hundreds of worn moccasins, which are of particular interest because of the Canadian-Subarctic style in which they are made, and for their potential to reveal more about the hunting strategies of their wearers. Here, we isolate ancient DNA from 38 Promontory Cave 1 moccasin and hide fragments and use these data to determine the animal species and sex used to construct the moccasins. We found that moccasins were all made from bison and most (87%) were females. The strong female bias in our data, which we demonstrate is a significant departure from sex ratios in present-day bison herds, suggests that the occupants of the cave were purposefully targeting female bison for moccasin manufacture. Our study is the first to our knowledge to determine faunal sex ratios from an assemblage of archaeological leather and highlights another potential avenue for ancient DNA technologies to augment what can be learned from the archaeological record.